Objective: To evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.
Methods: Direct solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.
Results: The total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.
Conclusion: Hot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.